First published online August 31, 2006
Stem Cells
Vol. 24 No.
12
December 2006, pp.
2888
-2890
doi:10.1634/stemcells.2006-0387; www.StemCells.com
© 2006 AlphaMed Press
TRANSLATIONAL AND CLINICAL RESEARCH |
Cell Culture Medium Composition and Translational Adult Bone Marrow-Derived Stem Cell Research
Marc Gabriel Berger,
Richard Veyrat-Masson,
Chantal Rapatel,
Stéphane Descamps,
Jacques Chassagne,
Nathalie Boiret-Dupre
CHU Clermont-Ferrand, GECOM, Hématologie Biologique, Hôtel-Dieu, Boulevard Léon Malfreyt, 63058 Clermont-Ferrand Cedex 1, France
Key Words. Clinical translation • Mesenchymal stem cells • Ex vivo expansion • Culture
Correspondence: Marc G. Berger, M.D., Ph.D., Groupe d'Etude des Cellules d'Origine Médullaire (GECOM), Hématologie Biologique, Hôtel-Dieu, Boulevard Léon Malfreyt, 63058 Clermont-Ferrand Cedex 1, France. Telephone: 33 4 73 75 06 82; Fax: 33 4 73 75 06 83; e-mail: mberger{at}chu-clermontferrand.fr
Received June 26, 2006;
accepted for publication August 22, 2006.
First published online in STEM CELLS EXPRESS August 31, 2006.
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ABSTRACT
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For most therapeutic strategies using MSC, the preliminary amplification is carried out in media containing fetal calf serum (FCS). The theoretical health risk of using a xenogenic serum, a recent practice for which we have limited data, cannot be underestimated, while amplification using human serum (HS) remains controversial. At present, the available information on multipotentiality, self-renewal, and transplantability does not permit the selection of FCS rather than HS. Cellular modifications observed during cell passage seem to indicate a gradual impairment of cells in relation to native MSC, suggesting the making of short cell cultures without necessarily trying to reinfuse a high number of MSC in patients. With this approach, the volume of HS required would remain limited. While clinical studies have already started, many problems remain, such as evaluating the quality of the initial mesenchymal compartment and the biological properties of the cell suspension with FCS compared to those with HS, and depending on culture time.
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INTRODUCTION
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We have followed with great interest the discussion between P.A. Sotiropoulou's team and Drs. Dimarakis and Levicar [1, 2]. The elaboration of a culture medium adapted to the production of MSC—or more exactly, given current knowledge, of a population of adherent cells containing multipotent mesenchymal cells—for the clinical application of cell therapy remains a crucial matter. Since, at the moment, a serum-free medium without growth factors is not capable of amplifying these cells in vitro, the type of serum used (fetal calf serum (FCS) or human serum (HS), allogeneic or autologous, serum or plasma) is a critical issue. Although clinical studies with MSC cultured with FCS have not shown any marked secondary effect, the possible contamination (prion, virus, zoonosis) or immunological reaction against xenogenic serum antigens, have not been investigated, or are difficult to identify, and therefore we should proceed with caution. Moreover, follow-up is too limited to have any long-term assessment of the consequences [3–5]. The study performed by J.C. Chachques et al. [6] suggests that the ex vivo culture phase induces subtle and unexpected modifications in the biology of amplified MSC when the cell product is released and infused into the patient. In theory, using HS, in particular autologous serum, should eliminate or reduce the risk of these secondary effects. Different considerations indicate that we should not rule out the possibility of using HS, especially autologous serum.
Comparative studies of HS and FCS are not so precise. Most agree in recognizing that from the first passage, autologous HS induces a greater amplification than does FCS [7–11]. However, it is currently accepted that the life span of MSC is limited in vitro, replicative senescence occurring between approximately 25–30 population doublings (PD) [12]. This phenomenon is related to a shortening of telomeres, as has been described for other systems [13–15]. Also, to really compare MSC produced with two types of sera, it would be necessary to compare cells obtained after the equivalent number of PD. The other parameters of cell culture, particularly initial cellular density, basic medium, and the presence of any cytokines, are variables that may explain, at least to some extent, the inconsistencies between study results [8, 10, 16].
Certain factors such as the decrease in multiplication speed, progenitor frequency, telomere length, and multilineage differentiation strongly suggest that cells are impaired when amplified in FCS-containing media, in relation to fresh, non-manipulated cells, and study of these native cells seems essential. One approach may be to consider that the most relevant criterion for choosing a serum should be its ability to obtain MSC with properties as close as possible to those of native MSC. This implies that we have to resolve two problems: (a) identifying progenitors in fresh bone marrow, which involves quality control of the mesenchymal compartment of non-manipulated grafts [17] and (b) evaluating the self-renewal capacity of MSC with suitable tools.
One argument given by P.A. Sotiropoulou et al. for using FCS is the limited amount of available autologous serum. This should be discussed, bearing in mind several factors. (a) The idea that cells need to be amplified before therapeutic use is based on a doubtful rationale: the need to amplify cells because they are rare in bone marrow. Nothing proves that the daughter cells produced by native MSC compensate by their numbers the properties lost or modified during amplification. Conversely, with an allogeneic strategy, amplification remains of great interest for purifying MSC and eliminating the hematopoietic cells involved in the host-graft relation. It is likely that they should be purified by short in vitro amplification, while retaining their properties. Needs for serum would thus be reduced. (b) Culture conditions could be adapted to clinical application. For example, bone reconstruction is a cell therapy, which appears particularly attractive today, from the public health perspective. In this case, the number of required progenitors could be limited and HS would be of particular interest since it seems to favor osteoblast differentiation [8, 18, 19]. (c) In most therapeutic protocols, the infusion of cells can be programmed and serum collection scheduled. (d) In the literature, the results for allogeneic serum are contradictory. Some groups have observed interesting proliferation [7, 20], and others have observed very rapid senescence [10, 21]. Donor characteristics (i.e., age) may play a key role and could be particularly useful in orthopedics [22, 23]. (e) Moreover, the most primary phenotypes have been described in poor, even serum-free culture media [24, 25], and so it seems possible that low serum concentrations, while limiting cell proliferation, may enable production of cells for therapeutic use. This justifies further study.
To conclude, although clinical studies have already started, many questions remain unsolved. We consider it essential to assess the benefit of HS, autologous and heterologous, for purification and/or amplification of MSC, depending on the clinical application. Relying on the experience we have with hematopoietic stem cells for cell therapy, we must now define a quality control for bone marrow grafts, identify native mesenchymal progenitors, and evaluate the effects of amplification to assess both autologous and allogeneic HS in comparison with FCS to make an informed choice.
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DISCLOSURES
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The authors indicate no potential conflicts of interest.
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Abstract
Introduction
